Day 29 and 30

After my OGD plates were incubated for 1 and a half hours I treated them with 0.3, 1 and 3uM of TAT and TAT-p110 as follows:


Calculation of the volumes of each of the diluted peptides required to make up final concentration of 0.3 Μm in 500 μl media:

Use the equation V = n/C

For TAT (10mM stock solutions):

To make 0.3μM final: Use the 0.1mM dilution tube, ie. 1/100. 0.1mM is the same as 100μM.
Therefore: 0.3 μM/100 μM = 3x10-3.
3x10-3 x 500 μl = 1.5 μl/500 μl media in ONE WELL. For 15 wells: 1.5 μl / 500 μl x 15 = 22.5 μl/7.5 ml media.
To make 1 μM final: Use 1mM dilution tube, ie. 1/10. 1mM is the same as 1000 μM.
Therefore: 1 μM/1000 μM = 1x10-3.
1x10-3 x 500 μl = 0.5 μl/500 μl media in ONE WELL.
For 15 wells: 0.5 μl / 500 μl x 15 = 7.5 μl/7.5ml media

To make 3 μM final: Use 1mM dilution tube, ie. 1/10. 1mM is the same as 1000 μM.
Therefore: 3 μM/1000 μM = 3x10-3.
3x10-3 x 500 μl = 1.5 μl/500 μl media in ONE WELL. For 15 wells: 1.5 μl / 500 μl x 15 = 22.5 μl/7.5ml media.

For TAT-p110 (4mM Stock solution):
 To make 0.3μM final: Use the 0.04mM dilution tube, ie. 1/100. 0.04mM is the same as 40μM.
Therefore: 0.3 μM/40 μM = 7.5x10-3
7.5x10-3 x 500 μl = 3.75 μl/500 μl media in ONE WELL. For 15 wells: 3.75 μl / 500 μl x 15 = 56.25 μl/2.5ml media

To make 1 μM final: Use 0.4mM dilution tube, ie. 1/10. 0.4mM is the same as 400 μM.
Therefore: 1 μM/400 μM = 2.5x10-3.
2.5x10-3 x 500 μl = 1.25 μl/500 μl media in ONE WELL. For 15 wells: 1.25 μl /500 μl x 15 = 18.75 μl/7.5ml media

To make 3 μM final: Use 0.4mM dilution tube, ie. 1/10. 0.4mM is the same as 400 μM.
Therefore: 3 μM/400 μM = 7.5x10-3.
7.5x10-3 x 500 μl = 3.75 μl/500 μl media in ONE WELL. For 15 wells: 3.75 μl / 500 μl x 15 = 56.25 μl/2.5ml media
After incubating the plates overnight with the drugs i assayed them the next day:
After my assays, I could finally conduct confocal microscopy on my slides! Here is a sneak peak of the confocals from the Leica programme...




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